A. Field of the Invention
This invention is directed to methods for the enhanced production and recovery of taxol, baccatin III and other taxanes by cell cultures of Taxus species.
B. Related Art
The Taxane Supply Challenge
Taxol is a diterpenoid alkaloid originally isolated from the bark of the pacific yew, Taxus brevifolia (Wani, et al. 1971, J. Am. Chem. Soc., 93, 2325-2327). Interest in taxol began when the National Cancer Institute (NCI), in a large-scale screening program, found that crude bark extracts exhibited anti-tumor activities. Since then, clinical trials have confirmed that taxol is extremely effective against refractory ovarian cancers, and against breast and other cancers. Taxol has been pronounced as a breakthrough in chemotherapy because of its fundamentally different mechanism of cytotoxicity, i.e., by inhibiting depolymerization of microtubules (see Rowinsky, et al., 1990, J. Natl. Cancer Inst., 82, 1247-1259).
A daunting variable in the taxol equation has been supply. Bark-derived taxol has been discontinued as the primary source of commercial drug; large-scale production has been achieved by semi-synthesis, i.e., chemical attachment of a side chain to the plant-derived precursor, 10-deacetylbaccatin III. Total synthesis, while accomplished by academic laboratories, shows little promise as a viable commercial route to taxol. There is therefore an urgent need to develop cost-effective, environmentally-benign, and consistent sources of supply to keep up with the growing demand for taxol.
In addition to taxol, there is an urgent need to develop processes for the commercial production of related taxane molecules. Derivatives of taxol such as Taxotere have already been introduced into the world market. Further, tremendous research activity is being focused on the discovery and development of novel taxane derivatives with advantageous activity. These advances are likely to create an ongoing need for large quantities of an appropriate starting “skeleton” molecule from which any given derivative could be effectively synthesized.
One example of such a molecule is the aforementioned precursor, 10-deacetylbaccatin III, which is used as the starting point for semi-synthetic taxol. Another desirable starting molecule for semi-synthetic production of taxol and other derivatives is baccatin III. Baccatin III is normally not accumulated as a major taxane in planta, and hence there is no facile large-scale natural source for this molecule. However, it is a very desirable starting point for semi-synthesis because of its chemical closeness to taxol; for example, the steps that are required for acetylation of the 10 position of 10-deacetylbaccatin III are circumvented if baccatin III is the starting point rather than 10-deacetylbaccatin III.
This invention is related to the development of plant cell culture-based processes for the commercial production of taxol, baccatin III and other taxanes.
Tissue Cultures as a Source of Plant-Derived Chemicals
The ability of plant cells to divide, grow, and produce secondary metabolites under a variety of different cultural regimes has been amply demonstrated by a number of groups. At present, two compounds, shikonin (a red dye and anti-inflammatory) and ginsengoside (a tonic in oriental medicine) are produced by tissue-culture processes in Japan. Many other processes are reportedly close to commercialization, including vanillin, berberine and rosmarinic acid (see Payne, et al. 1991, “Plant Cell and Tissue Culture in Liquid Systems,” Hanser Publishers, Munich).
The advantages of a plant cell culture process for taxol, baccatin III, and taxanes are many: (i) A cell culture process ensures a limitless, continuous and uniform supply of product, and is not subject to pests, disasters and seasonal fluctuations, (ii) cell cultures can be cultivated in large bioreactors, and can be induced to overproduce the compound of interest by manipulating environmental conditions, (iii) cell cultures produce a simpler spectrum of compounds compared to bark or needles, considerably simplifying separation and purification, (iv) a cell culture process can adapt quickly to rapid changes in demand better than agriculture-based processes, (v) besides supplying taxol, baccatin III or other precursors, a cell culture process could also produce taxane compounds that exhibit advantageous bioactivity profiles, or that could be converted into other bioactive derivatives.
Since aseptic, large-scale, plant cell cultivation is inherently expensive, a cell culture process becomes commercially relevant only when these costs are offset by high productivity. Every plant species and target metabolite is different, and different approaches are necessary for every particular system. This invention focuses on creative and skilled approaches for obtaining highly productive plant cell cultures for taxol, baccatin III, and taxane production.
Problems with Tissue Cultures of Woody Plants and Conifers
A historical survey of the literature suggests that whereas herbaceous plants have been relatively easily manipulated in culture, productive cultures of woody plants and conifers have been achieved only with difficulty.
The growth of secondary metabolite producing gymnosperm- and conifer-cultures have been generally low. For example, Berlin and Witte, (1988, Phytochemistry, 27, 127-132) found that cultures of Thuja occidentalis increased their biomass by only ca. 30% in 18 days. Van Uden et al. (1990, Plant Cell Reports, 9, 257-260) reported a biomass increase of 20-50% in 21 days for suspensions of Callitris drummondii. Westgate et al. (1991, Appl. Microbiol. Biotechnol., 34, 798-803) reported a doubling time of ca. 10 days for suspensions of the gymnosperm, Cephalotaxus harringtonia. As summarized by Bornman (1983, Physiol. Plant. 57, 5-16), a tremendous amount of effort has been directed towards medium development for spruce suspensions (Picea abies). This collective work demonstrates that gymnosperm suspensions are indeed capable of rapid growth, but that no generalities can be applied, and that media formulations for different cell lines must be optimized independently.
A survey of secondary metabolite productivity among gymnosperm cultures also points to the difficulty of inducing rapid biosynthesis compared to herbaceous species. For example, cultures of Cephalotaxus harringtonia produced terpene alkaloids at a level of only 1% to 3% of that found in the parent plant (Delfel and Rothfus, 1977, Phytochemistry, 16, 1595-1598). Even upon successful elicitation, Heinstein (1985, Journal of Natural Products, 48, 1-9) was only able to approach the levels produced in the parent plant (ca. 0.04% dry weight total alkaloids). Van Uden et al (1990) were able to induce suspension cultures of the conifer Callitris drummondii to produce podophyllotoxin, but only at levels one tenth of that produced by the needles. The ability of Thuja occidentalis to produce significant levels of monoterpenes (10-20 mg/L) and the diterpenoid dehydroferruginol (2-8 mg/L) has been convincingly demonstrated by Berlin et al. (1988). However, these results were obtained with a slow-growing (30% biomass increase in 18 days) and low cell density (5 to 7 grams dry weight per liter) culture.
Cell Culture for Taxane Production
The difficulties in achieving rapid growth and high productivity encountered in gymnosperm-suspensions have generally been reflected in the reports so far on taxane production in Taxus cell cultures.
Jaziri et al. (1991, J Pharm. Belg., 46, 93-99) recently initiated callus cultures of Taxus baccata, but were unable to detect any taxol using their immunosorbent assay. Wickremesinhe and Arteca (1991, Plant Physiol., 96, (Supplement) p. 97) reported the presence of 0.009% dry weight taxol in callus cultures of Taxus media (cv. hicksii), but details on the doubling times, cell densities, and the time-scale over which the reported taxol was produced, were not indicated.
U.S. Pat. No. 5,019,504 (Christen et al. 1991) describes the production and recovery of taxane and taxane-like compounds by cell cultures of Taxus brevifolia. These workers reported taxol production at a level of 1 to 3 mg/L in a two- to four-week time frame. They also reported a cell mass increase of “5-10 times in 3-4 weeks”, which corresponds to doubling times of ca. 7 to 12 days.
Significant increases in taxane titers and volumetric productivity are required before an economically-viable plant cell culture process for taxane production can supply the projected annual demand of many hundreds of kilograms per year.